1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives as immunomodulators

ABSTRACT

The present invention relates to synthetic methods for 1,3,4-oxadiazole and 1,3,4-thiadiazole compounds. The general synthetic scheme is: 
                         
R 1  substituents are —CH 2 OH, —CH(CH 3 )OH, —CH 2 Ph, —CH 3 , or —CH 2 C(O)NH 2  optionally substituted with alkyl or aralkyl. R 3  substituents are —CH 2 OH, —CH 3 , —CH 2 CH 2 C(O)OH, —CH 2 CH 2 C(O)NH 2 , —CH 2 C(O)NH 2 , or —CH 2 C(O)OH, wherein the carboxylic acids and amides are optionally substituted with alkyl or aralkyl.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of pending U.S.Ser. No. 15/296,292, filed Oct. 18, 2016, which is a continuation under35 U.S.C. § 120 of application U.S. Ser. No. 14/916,290, filed Mar. 3,2016, now abandoned, which is a national stage application under 35U.S.C. § 371 of international application PCT/IB2014/064281, filed onSep. 5, 2014, now abandoned, which claims the benefit of priority toIndian provisional application number 4012/CHE/2013, filed Sep. 6, 2013,now abandoned, and for which a certified copy thereof is found in thefile wrapper of U.S. Ser. No. 14/916,290, the contents of all of whichare hereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION Technical Field

The present invention relates to 1,3,4-oxadiazole and 1,3,4-thiadiazolecompounds therapeutically useful as immune modulators. The inventionalso relates to pharmaceutical compositions comprising the said1,3,4-oxadiazole and 1,3,4-thiadiazole compounds as therapeutic agents.

Description of the Related Art

Programmed cell death-1 (PD-1) is a member of the CD28 superfamily thatdelivers negative signals upon interaction with its two ligands, PD-L1or PD-L2. PD-1 and its ligands are broadly expressed and exert a widerrange of immunoregulatory roles in T cells activation and tolerancecompared with other CD28 members. PD-1 and its ligands are involved inattenuating infectious immunity and tumor immunity, and facilitatingchronic infection and tumor progression. The biological significance ofPD-1 and its ligand suggests the therapeutic potential of manipulationof PD-1 pathway against various human diseases (Ariel Pedoeem et al.,Curr Top Microbiol Immunol. (2011); 350:17-37).

T-cell activation and dysfunction relies on direct and modulatedreceptors. Based on their functional outcome, co-signaling molecules canbe divided as co-stimulators and co-inhibitors, which positively andnegatively control the priming, growth, differentiation and functionalmaturation of a T-cell response (Li Shi, et al., Journal of Hematology &Oncology 2013, 6:74).

Therapeutic antibodies that block the programmed cell death protein-1(PD-1) immune checkpoint pathway prevent T-cell down regulation andpromote immune responses against cancer. Several PD-1 pathway inhibitorshave shown robust activity in various phases of clinical trials (R DHarvey, Clinical Pharmacology & Therapeutics (2014); 96 2, 214-223).

Programmed death-1 (PD-1) is a co-receptor that is expressedpredominantly by T cells. The binding of PD-1 to its ligands, PD-L1 orPD-L2, is vital for the physiological regulation of the immune system. Amajor functional role of the PD-1 signaling pathway is the inhibition ofself-reactive T cells, which serve to protect against autoimmunediseases. Elimination of the PD-1 pathway can therefore result in thebreakdown of immune tolerance that can ultimately lead to thedevelopment of pathogenic autoimmunity. Conversely, tumor cells can attimes co-opt the PD-1 pathway to escape from immunosurveillancemechanisms. Therefore, blockade of the PD-1 pathway has become anattractive target in cancer therapy. Current approaches include sixagents that are either PD-1 and PD-L1 targeted neutralizing antibodiesor fusion proteins. More than forty clinical trials are underway tobetter define the role of PD-1 blockade in variety of tumor types.(Hyun-Tak Jin et al., Clinical Immunology (Amsterdam, Netherlands)(2014), 153(1), 145-152).

International applications WO 01/14557, WO 02/079499, WO 2002/086083, WO03/042402, WO 2004/004771, WO 2004/056875, WO2006121168, WO2008156712,WO2010077634, WO2011066389, WO2014055897, WO2014059173, WO2014100079 andU.S. Pat. No. 8,735,553 report PD-1 or PD-L1 inhibitory antibodies orfusion proteins.

Further, International applications, WO2011161699, WO2012/168944,WO2013144704 and WO2013132317report peptides or peptidomimetic compoundswhich are capable of suppressing and/or inhibiting the programmed celldeath 1 (PD1) signaling pathway.

Still there is a need for more potent, better and/or selective immunemodulators of PD-1 pathway. The present invention provides1,3,4-oxadiazole and 1,3,4-thiadiazole compounds which are capable ofsuppressing and/or inhibiting the programmed cell death 1 (PD1)signalling pathway.

SUMMARY OF INVENTION

In accordance with the present invention, 1,3,4-oxadiazole and1,3,4-thiadiazole compounds or a pharmaceutically acceptable salt or astereoisomer thereof, provided which are capable of suppressing and/orinhibiting the programmed cell death 1 (PD1) signalling pathway.

In one aspect, the present invention provides a 1,3,4-oxadiazole and1,3,4-thiadiazole compounds of formula (I):

wherein,

R₁ is side chain of an amino acid selected from Ser, Thr, Phe, Ala orAsn;

X is S or O;

R₂ is hydrogen or —CO-Aaa;

Aaa is an amino acid residue selected from Ser, Asn or Thr; wherein aC-terminus thereof is a free terminus, is amidated or is esterified;

R₃ is side chain of an amino acid selected from Ser, Ala, Glu, Gln, Asnor Asp;

is an optional bond;

R₄ and R₅ independently are hydrogen or absent;

or a pharmaceutically acceptable salt or a stereoisomer thereof.

In a further aspect of the present invention, it relates to thepharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt or a stereoisomer and processes forpreparing thereof.

In yet another aspect of the present invention, it provides use of1,3,4-oxadiazole and 1,3,4-thiadiazole compounds of formula (I) or apharmaceutically acceptable salt or a stereoisomer thereof, which arecapable of suppressing and/or inhibiting the programmed cell death 1(PD1) signaling pathway.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides 1,3,4-oxadiazole and 1,3,4-thiadiazolecompounds as therapeutic agents useful for treatment of disorders viaimmunopotentiation comprising inhibition of immunosuppressive signalinduced due to PD-1, PD-L1, or PD-L2 and therapies using them.

Each embodiment is provided by way of explanation of the invention, andnot by way of limitation of the invention. In fact, it will be apparentto those skilled in the art that various modification and variations canbe made in the present invention without departing from the scope orspirit of the invention. For instance, features illustrated or describedas part of one embodiment can be used on another embodiment to yield astill further embodiment. Thus it is intended that the present inventioncover such modifications and variations as come within the scope of theappended claims and their equivalents. Other objects, features, andaspects of the present invention are disclosed in, or are obvious from,the following detailed description. It is to be understood by one ofordinary skill in the art that the present discussion is a descriptionof exemplary embodiments only, and is not to be construed as limitingthe broader aspects of the present invention.

In one embodiment, the present invention relates to compounds of formula(I)

wherein,

R₁ is side chain of an amino acid selected from Ser, Thr, Phe, Ala orAsn;

X is S or O;

R₂ is hydrogen or —CO-Aaa;

Aaa is an amino acid residue selected from Ser, Asn or Thr; wherein aC-terminus thereof is a free terminus, is amidated or is esterified;

R₃ is side chain of an amino acid selected from Ser, Ala, Glu, Gln, Asnor Asp;

is an optional bond;

R₄ and R₅ independently are hydrogen or absent;

or a pharmaceutically acceptable salt or a stereoisomer thereof.

In yet another embodiment, the present invention provides compounds offormula (IA)

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein,

R₁ is side chain of an amino acid selected from Ser, Thr, Phe, Ala orAsn;

X is S or O;

R₂ is hydrogen or —CO-Aaa;

R₃ is side chain of an amino acid selected from Ser, Ala, Glu, Gln, Asnor Asp;

Aaa is an amino acid residue selected from Ser, Asn or Thr; wherein aC-terminus thereof is a free terminus, is amidated or is esterified.

In yet another further embodiment, the present invention providescompounds of formula (IB)

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein,

R₁ is side chain of an amino acid selected from Ser, Thr, Phe, Ala orAsn;

R₃ is side chain of an amino acid selected from Ser, Ala, Glu, Gln, Asnor Asp;

Aaa is an amino acid residue selected from Ser, Asn or Thr; wherein aC-terminus thereof is a free terminus, is amidated or is esterified.

In yet another further embodiment, the present invention providescompounds of formula (IC)

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein,

R₁ is side chain of an amino acid selected from Ser, Thr, Phe, Ala orAsn;

R₃ is side chain of an amino acid selected from Ser, Ala, Glu, Gln, Asnor Asp;

Aaa is an amino acid residue selected from Ser, Asn or Thr; wherein aC-terminus thereof is a free terminus, is amidated or is esterified.

In yet another further embodiment, the present invention providescompounds of formula (I), wherein,

R₁ is side chain of Ser or Thr;

R₂ is —CO-Aaa;

Aaa is an amino acid residue Ser or Thr; wherein the C-terminus is free;

R₃ is side chain of Asn, Gln, Glu or Asp.

The embodiment below are illustrative of the present invention and arenot intended to limit the claims to the specific embodimentsexemplified.

According to one embodiment, specifically provided are compounds of theformula (I) and (IA), in which X is O.

According to another embodiment, specifically provided are compounds ofthe formula (I) and (IA) in which X is S.

According to yet another embodiment, specifically provided are compoundsof the formula (I) and (IA) in which R₂ is hydrogen.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₄ and R₅ are hydrogen.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₄ and R₅ are absent.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₂ is —CO-Ser.

According to yet another embodiment, specifically provided are compoundsof the formula (I) in which R₂ is —CO-Thr.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA), (IB) and (IC) in which R₁ is side chain ofSer.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA), (IB) and (IC) in which R₁ is side chain ofThr.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA) and (IC) in which R₁ is side chain of Phe, Alaor Asn.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA), (IB) and (IC) in which R₃ is side chain ofAsn.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA) and (IB) in which R₃ is side chain of Ser.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA) and (IC) in which R₃ is side chain of Gln.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA) and (IC) in which R₃ is side chain of Glu.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA) and (IC) in which R₃ is side chain of Ala orAsp.

According to yet another embodiment, specifically provided are compoundsof the formula (IB) and (IC) in which Aaa is Ser.

According to yet another embodiment, specifically provided are compoundsof the formula (IC) in which Aaa is Thr.

According to yet another embodiment, specifically provided are compoundsof the formula (I), (IA) and (IB) in which one, more or all amino acid/sis/are D amino acid/s.

In an embodiment, specific compounds of formula (I) without anylimitation are enumerated in Table (1).

TABLE 1 Com- pound No. Structure  1.

 2.

 3.

 4.

 5.

 6.

 7.

 8.

 9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

and 19.

20.

or a pharmaceutically acceptable salt thereof or a stereoisomer thereof.

The compounds as disclosed in the present invention are formulated forpharmaceutical administration.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising the compound as disclosed, and a pharmaceuticallyacceptable carrier or a diluent.

In another embodiment, the said pharmaceutical composition furthercomprising at least one of an anticancer agent, chemotherapy agent, orantiproliferative compound.

In one embodiment, the present invention provides the compounds asdisclosed in the present invention for use as a medicament.

In another embodiment, the present invention provides the compounds asdisclosed in the present invention for use as a medicament for thetreatment of cancer or infectious disease.

In another embodiment, the present invention provides the compounds asdisclosed in the present invention for use as a medicament for thetreatment bone cancer, cancer of the head or neck, pancreatic cancer,skin cancer, cutaneous or intraocular malignant melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, testicular cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease,non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, chronic oracute leukemias including acute myeloid leukemia, chronic myeloidleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,solid tumours of childhood, lymphocytic lymphoma, cancer of the bladder,cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasmof the central nervous system (CNS), primary CNS lymphoma, tumourangiogenesis, spinal axis tumour, brain stem glioma, pituitary adenoma,Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-celllymphoma, environmentally induced cancers including those induced byasbestos, and combinations of said cancers.

In another embodiment, the present invention provides the compounds asdisclosed in the present invention for use in the treatment of cancer.

In another embodiment, the present invention provides the compounds asdisclosed in the present invention for use in the treatment ofinfectious disease.

In one embodiment, the present invention provides the compounds asdisclosed in the present invention for use as a medicament for thetreatment of bacterial infectious disease, a viral infectious disease ora fungal infectious disease.

In one embodiment, the present invention provides a method of treatmentof cancer, wherein the method comprises administration of an effectiveamount of the compound of the present invention to the subject in needthereof.

In another embodiment the present invention provides a method ofmodulating an immune response mediated by PD-1 signaling pathway in asubject, comprising administering to the subject therapeuticallyeffective amount of the compound of the present invention such that theimmune response in the subject is modulated.

In yet another embodiment the present invention provides a method ofinhibiting growth of tumour cells and/or metastasis in a subject,comprising administering to the subject a therapeutically effectiveamount of compound of the present invention capable of inhibiting theprogrammed cell death 1 (PD1) signaling pathway.

The said tumour cells include cancer such as but not limited to bonecancer, cancer of the head or neck, pancreatic cancer, skin cancer,cutaneous or intraocular malignant melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer,testicular cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin'slymphoma, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, chronic or acute leukemiasincluding acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, solid tumours ofchildhood, lymphocytic lymphoma, cancer of the bladder, cancer of thekidney or ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumour angiogenesis, spinalaxis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers.

In yet another further embodiment the present invention provides amethod of treating an infectious disease in a subject comprisingadministering to the subject a therapeutically effective amount of thecompound of the present invention capable of inhibiting the programmedcell death 1 (PD1) signaling pathway such that the subject is treatedfor the infectious disease.

Still yet another embodiment of the present invention provides a methodof treating bacterial, viral and fungal infections in a subjectcomprising administering to the subject a therapeutically effectiveamount of the compound of the present invention capable of inhibitingthe programmed cell death 1 (PD1) signalling pathway such that thesubject is treated for the bacterial, viral and fungal infections.

The infectious disease includes but not limited to HIV, Influenza,Herpes, Giardia, Malaria, Leishmania, the pathogenic infection by thevirus Hepatitis (A, B, & C), herpes virus (e.g., VZV, HSV-I, HAV-6,HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus,flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus andarboviral encephalitis virus, pathogenic infection by the bacteriachlamydia, rickettsial bacteria, mycobacteria, staphylococci,streptococci, pneumonococci, meningococci and conococci, Klebsiella,Proteus, Serratia, Pseudomonas, E. coli, Legionella, diphtheria,Salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague,leptospirosis, and Lyme's disease bacteria, pathogenic infection by thefungi Candida (albicans, krusei, glabrata, tropicalis, etc.),Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), GenusMucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomycesdermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis andHistoplasma capsulatum, and pathogenic infection by the parasitesEntamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoebasp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii,Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosomacruzi, Leishmania donovani, Toxoplasma gondi, Nippostrongylusbrasiliensis.

The compounds of the present invention may be used as single drugs or asa pharmaceutical composition in which the compound is mixed with variouspharmacologically acceptable materials.

The pharmaceutical composition is usually administered by oral orinhalation routes, but can be administered by parenteral administrationroute. In the practice of this invention, compositions can beadministered, for example, by orally, intravenous infusion, topically,intraperitoneally, intravesically or intrathecally. Examples of theparenteral administration includes but not limited to intraarticular (inthe joints), intravenous, intramuscular, intradermal, intraperitoneal,and subcutaneous routes, include aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. Oral administration, parenteraladministration, subcutaneous administration and intravenousadministration are the preferred methods of administration.

The dosage of the compounds of the present invention varies depending onage, weight, symptom, therapeutic efficacy, dosing regimen and/ortreatment time. Generally, they may be administered by oral orinhalation routes, in an amount of 1 mg to 100 mg per time, from once acouple of days, once 3 days, once 2 days, once a day to a couple oftimes a day, in the case of an adult, or continuously administered byoral or inhalation routes from 1 to 24 hours a day. Since the dosage isaffected by various conditions, an amount less than the above dosage maysometimes work well enough, or higher dosage may be required in somecases.

The compounds of the present invention may be administered incombination with other drugs for (1) complementation and/or enhancementof prevention and/or therapeutic efficacy of the preventive and/ortherapeutic drug of the present invention, (2) dynamics, absorptionimprovement, dosage reduction of the preventive and/or therapeutic drugof the present invention, and/or (3) reduction of the side effects ofthe preventive and/or therapeutic drug of the present invention.

A concomitant medicine comprising the compounds of the present inventionand other drug may be administered as a combination preparation in whichboth components are contained in a single formulation, or administeredas separate formulations. The administration by separate formulationsincludes simultaneous administration and administration with some timeintervals. In the case of the administration with some time intervals,the compound of the present invention can be administered first,followed by another drug or another drug can be administered first,followed by the compound of the present invention. The administrationmethod of the respective drugs may be the same or different.

The dosage of the other drug can be properly selected, based on a dosagethat has been clinically used. The compounding ratio of the compound ofthe present invention and the other drug can be properly selectedaccording to age and weight of a subject to be administered,administration method, administration time, disorder to be treated,symptom and combination thereof. For example, the other drug may be usedin an amount of 0.01 to 100 parts by mass, based on 1 part by mass ofthe compound of the present invention. The other drug may be acombination of two or more kind of arbitrary drugs in a properproportion. The other drug that complements and/or enhances thepreventive and/or therapeutic efficacy of the compound of the presentinvention includes not only those that have already been discovered, butthose that will be discovered in future, based on the above mechanism.

Diseases on which this concomitant use exerts a preventive and/ortherapeutic effect are not particularly limited. The concomitantmedicine can be used for any diseases, as long as it complements and/orenhances the preventive and/or therapeutic efficacy of the compound ofthe present invention.

The compound(s) of the present invention can be used with an existingchemotherapeutic concomitantly or in a mixture form. Examples of thechemotherapeutic include an alkylation agent, nitrosourea agent,antimetabolite, anticancer antibiotics, vegetable-origin alkaloid,topoisomerase inhibitor, hormone drug, hormone antagonist, aromataseinhibitor, P-glycoprotein inhibitor, platinum complex derivative, otherimmunotherapeutic drugs and other anticancer drugs. Further, it can beused with a cancer treatment adjunct, such as a leucopenia (neutropenia)treatment drug, thrombocytopenia treatment drug, antiemetic and cancerpain intervention drug, concomitantly or in a mixture form.

In one embodiment, the compound(s) of the present invention can be usedwith other immunomodulators and/or a potentiating agent concomitantly orin a mixture form. Examples of the immunomodulator include variouscytokines, vaccines and adjuvants. Examples of these cytokines, vaccinesand adjuvants that stimulates immune responses include but not limitedto GM-CSF, M-CSF, G-CSF, interferon-α, β, or γ, IL-1, IL-2, IL-3, IL-12,Poly (I:C) and C_(p)G.

In another embodiment, the potentiating agents includes cyclophosphamideand analogs of cyclophosphamide, anti-TGFβ and Imatinib (Gleevac), amitosis inhibitor, such as paclitaxel, Sunitinib (Sutent) or otherantiangiogenic agents, an aromatase inhibitor, such as letrozole, an A2aadenosine receptor (A2AR) antagonist, an angiogenesis inhibitor,anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18antagonists.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in artto which the subject matter herein belongs. As used herein, thefollowing definitions are supplied in order to facilitate theunderstanding of the present invention.

As used herein, the term ‘compound(s)’ refers to the compounds disclosedin the present invention.

As used herein, the term “comprise” or “comprising” is generally used inthe sense of include, that is to say permitting the presence of one ormore features or components.

As used herein, the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

As used herein, the term “amino” refers to —NH₂ group. Unless set forthor recited to the contrary, all amino groups described or claimed hereinmay be substituted or unsubstituted.

As used herein, the term “amino acid” refers to amino acids having L orD stereochemistry at the alpha carbon.

“Pharmaceutically acceptable salt” is taken to mean an activeingredient, which comprises a compound of the formula (I) in the form ofone of its salts, in particular if this salt form imparts improvedpharmacokinetic properties on the active ingredient compared with thefree form of the active ingredient or any other salt form of the activeingredient used earlier. The pharmaceutically acceptable salt form ofthe active ingredient can also provide this active ingredient for thefirst time with a desired pharmacokinetic property which it did not haveearlier and can even have a positive influence on the pharmacodynamicsof this active ingredient with respect to its therapeutic efficacy inthe body.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic, andneither biologically nor otherwise undesirable and includes that whichis acceptable for veterinary as well as human pharmaceutical use.

The term “stereoisomer” refers to any enantiomers, diastereoisomers, orgeometrical isomers of the compounds of formula (I), wherever they arechiral or when they bear one or more double bond. When the compounds ofthe formula (I) and related formulae are chiral, they can exist inracemic or in optically active form. Since the pharmaceutical activityof the racemates or stereoisomers of the compounds according to theinvention may differ, it may be desirable to use the enantiomers. Inthese cases, the end product or even the intermediates can be separatedinto enantiomeric compounds by chemical or physical measures known tothe person skilled in the art or even employed as such in the synthesis.In the case of racemic amines, diastereomers are formed from the mixtureby reaction with an optically active resolving agent. Examples ofsuitable resolving agents are optically active acids such as the R and Sforms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid,mandelic acid, malic acid, lactic acid, suitable N-protected amino acids(for example N-benzoylproline or N-benzenesulfonylproline), or thevarious optically active camphorsulfonic acids. Also advantageous ischromatographic enantiomer resolution with the aid of an opticallyactive resolving agent (for example dinitrobenzoylphenylglycine,cellulose triacetate or other derivatives of carbohydrates or chirallyderivatised methacrylate polymers immobilised on silica gel).

The term “subject” includes mammals (especially humans) and otheranimals, such as domestic animals (e.g., household pets including catsand dogs) and non-domestic animals (such as wildlife).

“Therapeutically effective amount” or “efficient amount” refers tosufficient amount of the compound(s) of the present invention that (i)treats or prevents the particular disease, disorder or syndrome (ii)attenuates, ameliorates or eliminates one or more symptoms of theparticular disease, disorder or syndrome or (iii) prevents or delays theonset of one or more symptoms of the particular disease, disorder orsyndrome described herein. In the case of cancer, the therapeuticallyeffective amount of the drug may decrease the number of cancer cells;decrease the cancer size; inhibit (i.e., slow to some extent andalternatively stop) cancer cell infiltration into peripheral organs;suppress (i.e., slow to some extent and alternatively stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Inthe case of infectious disease states, the therapeutic effective amountis an amount sufficient to decrease or alleviate an infectious diseases,the symptoms of an infections caused by bacterial, viral and fungal.

Naturally-occurring amino acids are identified throughout by theconventional three-letter abbreviations indicated in the below Table 2.

TABLE 2 (Amino acid codes) Name 3-letter code Asparagine Asn Asparticacid Asp Alanine Ala Glutamic acid Glu Glutamine Gln Phenylalanin PheSerine Ser Threonine Thr

The abbreviations used in the entire specification may be summarizedhereinbelow with their particular meaning.

° C. (degree Celsius); δ (delta); % (percentage); brine (NaCl solution);CH₂Cl₂/DCM (Dichloromethane); br s (Broad singlet); Cs₂CO₃ (Caesiumcarbonate); d (Doublet); DMF (Dimethyl formamide); DMSO (Dimethylsulphoxide); DMSO-d₆ (Deuterated DMSO); EDC.HCl/EDCI (1-(3-Dimethylaminopropyl)-3-carbodiimide hydrochloride); Et₂NH (Diethylamine); Fmoc(Fluorenylmethyloxycarbonyl chloride); g or gr (gram); H or H₂(Hydrogen); H₂O (Water); HOBt/HOBT (1-Hydroxy benzotriazole); HCl(Hydrochloric acid); h or hr (Hours); Hz (Hertz); HPLC (High-performanceliquid chromatography); I₂ (Iodine); K₂CO₃ (Potassium carbonate); LCMS(Liquid chromatography mass spectroscopy); MeOH (Methanol); mmol(Millimoles); M (Molar); μl (Microliter); mL (Milliliter); mg(Milligram); m (Multiplet); MHz (Megahertz); MS (ES) (Massspectroscopy-electro spray); min. (Minutes); Na (Sodium); NaHCO₃ (Sodiumbicarbonate); NH₂NH₂.H₂O (Hydrazine hydrate); NMM (N-methyl morpholine);Na₂SO₄ (Sodium sulphate); N₂ (Nitrogen); NMR (Nuclear magnetic resonancespectroscopy); PD-L1 (Programmed death-ligand 1); PD-L2 (Programmed celldeath 1 ligand 2); prep-HPLC/preparative HPLC (PreparativeHigh-performance liquid chromatography); S (Singlet); ^(t)Bu (tertiarybutyl); TEA/Et₃N (Triethyl amine); TLC (Thin Layer Chromatography); THF(Tetrahydrofuran); TIPS (Triisopropylsilane); TFA/CF₃COOH(Trifluoroacetic acid); t (Triplet); t_(R)=(Retention time); TPP(Triphenylphosphine); etc.

EXPERIMENTAL

An embodiment of the present invention provides the preparation ofcompounds of formula (I) according to the procedures of the followingexamples, using appropriate materials. Those skilled in the art willunderstand that known variations of the conditions and processes of thefollowing preparative procedures can be used to prepare these compounds.Moreover, by utilizing the procedures described in detail, one ofordinary skill in the art can prepare additional compounds of thepresent invention.

The starting materials are generally available from commercial sourcessuch as Sigma-Aldrich, USA or Germany; Chem-Impex USA; G.L. Biochem,China and Spectrochem, India.

Purification and Characterization of Compounds

Analytical HPLC method: Analytical HPLC was performed using on ZIC HILIC200 A° column (4.6 mm×250 mm, 5 μm), Flow rate: 1.0 mL/min. The elutionconditions used are: Buffer A: 5 mmol ammonium acetate, Buffer B:Acetonitrile, Equilibration of the column with 90% buffer B and elutionby a gradient of 90% to 40% buffer B during 30 min.

Preparative HPLC Method: Preparative HPLC was performed using on SeQuantZIC HILIC 200 A° column (10 mm×250 mm, 5 μm), Flow rate: 5.0 ml/min. Theelution conditions used are: Buffer A: 5 mmol ammonium acetate (adjustto pH-4 with Acetic Acid), Buffer B: Acetonitrile, Equilibration of thecolumn with 90% buffer B and elution by a gradient of 90% to 40% bufferB during 20 min.

LCMS was performed on AP1 2000 LC/MS/MS triple quad (Applied biosystems) with Agilent 1100 series HPLC with G1315 B DAD, using MercuryMS column or using Agilent LC/MSD VL single quad with Agilent 1100series HPLC with G1315 B DAD, using Mercury MS column or using ShimadzuLCMS 2020 single quad with Prominence UFLC system with SPD-20 A DAD.

Example 1 Synthesis of Compound 1

Step 1a:

Potassium carbonate (7.9 g, 57.39 mmol) and Methyl iodide (1.3 mL, 21.04mmol) were added to a solution of compound 1a (5.0 g, 19.13 mmol) in DMF(35 mL) and stirred at room temperature for 2 h. The completeness of thereaction was confirmed by TLC analysis. The reaction mixture waspartitioned between water and ethyl acetate. Organic layer was washedwith water, brine, dried over Na₂SO₄ and evaporated under reducedpressure to get 5.0 g of compound 1b (Yield: 96.1%). LCMS: 176.1(M−Boc)⁺.

Step 1b:

Hydrazine hydrate (7.2 mL) was added to a solution of compound 1b (5.0g, 18.16 mmol) in methanol (30 mL) and stirred at room temperature for 2h. The completeness of the reaction was confirmed by TLC analysis. Thereaction mixture was evaporated under reduced pressure, the residueobtained was partitioned between water and ethyl acetate. Organic layerwas washed with water, brine, dried over Na₂SO₄ and evaporated underreduced pressure to get 4.0 g of compound 1c (Yield: 80.0%). LCMS: 276.3(M+H)⁺.

Step 1c:

NMM (0.67 ml, 6.52 mmol) was slowly added to a stirred solution of 1c(1.2 g, 4.35 mmol), 1d (1.43 g, 4.35 mmol), HOBt (0.7 g, 5.22 mmol) andEDC.HCl (0.99 g, 5.22 mmol) in DMF (15 mL) at 0°. The reaction mixturewas stirred at room temperature for 12 h. The completeness of thereaction was confirmed by TLC analysis. The reaction was quenched withice and the solid precipitated was filtered and dried under vacuum toobtain 2.0 g of pure product 1e (Yield: 83.3%). LCMS: 591.5 (M+Na)⁺.

Step 1d:

To a stirred solution of 1e (1.5 g, 2.63 mmol) in dry THF (15.0 mL) andDMF (5.0 mL) triphenylphosphine (1.38 g, 5.27 mmol) and iodine (1.33 g,5.27 mmol) were added at 0° C. After the iodine was completelydissolved, Et₃N (1.52 mL, 10.54 mmol) was added to this reaction mixtureat ice cold temperature. Reaction mixture was allowed to attain roomtemperature and stirred for 4 h. The completeness of the reaction wasconfirmed by TLC analysis. The reaction was quenched with ice water andextracted with ethyl acetate. Organic layer was washed with saturatedsodium thiosulphate and brine solution. The separated Organic layer wasdried over Na₂SO₄ and evaporated under reduced pressure to get residue,which was further purified by silica gel column chromatography (eluent:30% ethyl acetate in hexane) to afford 0.8 g of compound 1f (Yield:55%). LCMS: 551.3 (M+H)⁺.

Step 1e:

Fmoc group was deprotected by the addition of diethylamine (20.0 mL) toa solution of compound 1f (0.8 g, 1.45 mmol) in CH₂Cl₂ (20.0 mL) at 0°C. The reaction was stirred at room temperature for 2 h. The resultingsolution was concentrated in vacuum to get a thick gummy residue. Thecrude compound was purified by neutral alumina column chromatography(eluent: 2% methanol in chloroform) to afford 0.38 g of compound 1g(Yield: 80.0%): LCMS: 329.4 (M+H)⁺.

Step 1f:

Compound 1g (0.38 g, 1.16 mmol), TEA (0.33 mL, 2.32 mmol) dissolved inDMF (10 mL) were added drop wise to a solution of 1h (0.55 g, 1.39 mmol)at 0° for urea bond formation and the mixture was stirred at roomtemperature for 2 h. The completeness of the reaction was confirmed byTLC analysis. The reaction was quenched with ice water, the solidprecipitated was filtered and dried under vacuum to get crude compound,which was further purified by silica gel column chromatography (eluent:0-35% ethyl acetate in hexane) to get 0.4 g of product 1i (Yield:59.7%). LCMS: 586.4 (M+H)⁺.

Step 1g:

To a solution of compound 1i (0.4 g, 0.68 mmol) in CH₂Cl₂ (5 m L),trifluoroacetic acid (5 mL) and catalytic amount of triisopropylsilanewere added and stirred at room temperature for 3 h to remove the acidsensitive protecting groups. The resulting solution was concentratedunder nitrogen and the solid material was purified by preparative HPLCmethod as described under experimental conditions (Yield: 0.05 g). LCMS:318.0 (M+H)⁺; HPLC: t_(R)=10.96 min.

Synthesis of Compound 1h (NO₂—C₆H₄—OCO-Thr(tBu)-O^(t)Bu)

To a solution of 4-nitrophenylchloroformate (4.79 g, 23.77 mmol) in DCM(25.0 mL) was added a solution of H-Thr(tBu)-OtBu (5.0 g, 21.61 mmol)TEA (6.2 mL, 43.22 mmol) in CH₂Cl₂ (25 mL) slowly at 0° C. and allowedto stir for 30 min. The completion of the reaction was confirmed by TLCanalysis. After completion of reaction it was diluted with DCM andwashed with 1.0 M of citric acid followed by 1.0 M sodium carbonatesolution. The organic layer was dried over Na₂SO₄ and evaporated underreduced pressure to afford crude compound 1h, which was further purifiedby silica gel column chromatography (eluent: 0-5% ethyl acetate inhexane) to get 3.0 g of product 1h. ¹H NMR (CDCl₃, 400 MHz): δ 1.17 (s,9H), 1.28 (d, 3H), 1.50 (s, 9H), 4.11 (m, 1H), 4.28 (m, 1H), 5.89 (d,1H), 7.37 (d, 2H), 8.26 (d, 2H).

Example 2 Synthesis of Compound 2

Step 2a:

NMM (1.8 mL, 18.15 mmol) was slowly added to a stirred solution of 1c(2.0 g, 7.26 mmol), 2d (4.3 g, 7.26 mmol), HOBt (1.17 g, 8.7 mmol) andEDC.HCl (1.66 g, 8.7 mmol) in DMF (15 mL) at 0°. The reaction mixturewas stirred at room temperature for 12 h. The completeness of thereaction was confirmed by TLC analysis. The reaction was quenched withice, the solid precipitated was filtered and dried under vacuum toafford 3.7 g of pure product 2e (Yield: 59.6%). LCMS: 854.4 (M+H)⁺.

Step 2b:

To a stirred solution of 2e (3.7 g, 4.33 mmol) dissolved in dry THF(25.0 mL) and DMF (10.0 mL), triphenylphosphine (2.28 g, 8.66 mmol) andiodine (2.2 g, 8.66 mmol) were added at 0° C. After the iodine wascompletely dissolved, Et₃N (2.5 mL, 17.32 mmol) was added at sametemperature. The reaction mixture was stirred at room temperature for 4h. The completeness of the reaction was confirmed by TLC analysis. Thereaction was quenched with ice water and extracted with ethyl acetate.The organic layer was washed with saturated sodium thiosulphate andbrine solution. The separated organic layer was dried over Na₂SO₄ andevaporated under reduced pressure, which was further purified by silicagel column chromatography (eluent: 30% ethyl acetate in hexane) to get2.0 g of compound 2f (Yield: 55%). LCMS: 858.4 (M+Na)⁺.

Step 2c:

Diethylamine (30.0 mL) was added to a solution of compound 2f (2.0 g,1.17 mmol) in CH₂Cl₂ (30.0 mL) at 0°. The reaction mixture was stirredat room temperature for 1 h. The resulting solution was concentrated invacuum to get a thick gummy residue. The crude compound was purified byneutral alumina column chromatography (eluent: 2% methanol inchloroform) to afford 1.0 g of compound 2g (Yield: 71.4%). LCMS: 614.5(M+H)⁺.

Step 2d:

Compound 2g (1.0 g, 1.63 mmol) and TEA (0.47 mL, 3.2 mmol) dissolved inDMF (10 m L) were added drop wise to a solution of 1h (0.7 g, 1.79 mmol)at 0° C. The reaction mixture was then allowed to reach room temperatureand continued the stirring for 2 h. The completeness of the reaction wasconfirmed by TLC analysis. The reaction was quenched with ice water, thesolid precipitated was filtered and dried under vacuum. The crudecompound obtained was further purified by silica gel columnchromatography (eluent: 0-30% ethyl acetate in hexane) to get 0.8 g ofproduct 2i (Yield: 57.1%). LCMS: 871.6 (M+H)⁺.

Step 2e:

To a solution of compound 2i (0.8 g, 0.92 mmol) in CH₂Cl₂ (6 m L),trifluoroacetic acid (6 mL) and catalytic amount of triisopropylsilanewere added and stirred at room temperature for 3 h. The resultingsolution was concentrated under nitrogen and the solid material waspurified by preparative HPLC method described under experimentalconditions (Yield: 0.065 g). HPLC: t_(R)=12.01 min.; LCMS: 361.34(M+H)⁺.

Example 3 Synthesis of Compound 3

Step 3a:

Lawesson's reagent (2.85 g, 7.03 mmol) was added to a solution ofcompound 2e (4 g, 4.68 mmol) in THF (40 mL) and stirred at 75° C. for 4h. The completeness of the reaction was confirmed by TLC analysis. Thereaction mixture was evaporated under reduced pressure and the obtainedresidue was partitioned between ice water and ethyl acetate. The organiclayer was washed with NaHCO₃ solution followed brine solution. Theorganic layer was dried over Na₂SO₄, filtered and evaporated underreduced pressure to get residue which was further purified by silica gelcolumn chromatography (eluent: 0-5% ethyl acetate in hexane) to afford2.7 g of compound 3a (Yield: 67.66%). LCMS: 852.3 (M+H)⁺.

Step 3b:

Fmoc group on compound 3a was deprotected by adding diethylamine (3.8mL) to the solution of compound 3a (1 g, 1.17 mmol) in CH₂Cl₂ (3.8 mL).The reaction mixture was stirred at room temperature for 30 min. Theresulting solution was concentrated in vacuum to get a thick gummyresidue. The crude compound was purified by neutral alumina columnchromatography (eluent: 0-50% ethyl acetate in hexane then 0-5% methanolin chloroform) to attain 0.62 g of compound 3b. LCMS: 630.5 (M+H)⁺.

Step 3c:

To a solution of compound 3b (0.6 g) in CH₂Cl₂ (7.5 mL), trifluoroaceticacid (2.5 mL) and catalytic amount of triisopropylsilane were added andstirred at room temperature for 3 h. The resulting solution wasconcentrated in vacuum to get 0.13 g of compound 3 which was purified bypreparative HPLC method described under experimental conditions. LCMS:232.3 (M+H)⁺.

Example 4 Synthesis of Compound 4

Step 4a:

The urea linkage was carried out by coupling of compound 3b (0.5 g, 7.9mmol) in THF (10 m L) at room temperature with compound 4e (0.34 g, 7.9mmol). The coupling was initiated by the addition of TEA (0.16 g, 15.8mmol) in THF (10 m L) and the resultant mixture was stirred at roomtemperature. After 12 h, THF was evaporated from the reaction mass, andpartitioned between water and ethyl acetate. The organic layer waswashed with water, brine, dried over Na₂SO₄ and evaporated under reducedpressure to yield 4a, which was further purified by silica gel columnchromatography (eluent: 0-50% ethyl acetate in hexane) to get 0.45 g ofproduct 4a (Yield: 61.64%). LCMS: 921.8 (M+H)⁺.

Step 4b:

To a solution of compound 4a (0.55 g) in methanol (20 mL), was added 10%Pd—C (0.15 g) under inert atmosphere. The mixture was stirred for 1 hunder H₂ atmosphere. The completion of the reaction was confirmed by TLCanalysis. The Pd—C catalyst was then removed by filtration through aCelite® pad and washed with 20 mL of methanol. The combined organicfiltrate on evaporation under reduced pressure resulted in the isolationof the product 4b (Yield: 0.42 g, 85.71%). LCMS: 831.5 (M+H)⁺.

Step 4c:

To a solution of compound 4b (0.2 g, 0.3 mmol) in CH₂Cl₂ (5 mL),trifluoroacetic acid (5 mL) and catalytic amount of triisopropylsilanewere added and stirred at room temperature for 3 h. The resultingsolution was concentrated in vacuum and the solid material was purifiedby preparative HPLC method described under experimental conditions(Yield: 0.065 g). HPLC: t_(R)=14.1 min.; LCMS: 377.3 (M+H)⁺.

Synthesis of Compound 4e, (NO₂—C₆H₄—OCO-Thr(O^(t)Bu)-Bzl)

To a solution of compound Fmoc-Thr(^(t)Bu)-OH (15 g, 37.73 mmol) in 100mL of DMF, Cs₂CO₃ (14.75 g, 45.2 mmol) was added and the resultingmixture was cooled to 0° C. To the cooled reaction mixture benzylbromide (7.74 g, 45.2 mmol) was added and the solution was stirred atice cold temperature for 30 min and then at room temperature for 12 h.The reaction mixture was concentrated under reduced pressure and dilutedwith ethyl acetate. The organic layer was washed with water followed bybrine solution and dried over Na₂SO₄. The filtered solution wasconcentrated and purified by silica gel column chromatography (eluent:0-30% ethyl acetate in hexane) to get 18 g of 4c as white solid. LCMS:433.1 (M−OtBu)⁺, 397.2 (M−OBzl)⁺.

Fmoc group on compound 4c (25 g, 51.3 mmol) was deprotected by addingdiethylamine (100 mL) to compound 4d (25 g, 51.3 mmol) in CH₂Cl₂ (100mL) for 1 h with stirring at room temperature. The resulting solutionwas concentrated in vacuum and the thick residue was purified by neutralalumina column chromatography (eluent: 0-50% ethyl acetate in hexanethen 0-5% methanol in chloroform) to afford 10.6 g of compound 4d. LCMS:266.5 (M+H)⁺.

To a solution of compound 4d (1.5 g, 5.65 mmol) in CH₂Cl₂ (25 m L) wasadded TEA (1.14 g, 11.3 mmol) and the solution was stirred at roomtemperature for 5-10 min. To this mixture a solution of 4-nitrophenylchloroformate (1.4 g, 6.78 mmol) in CH₂Cl₂ (10 mL) was added and theresultant mixture was stirred at room temperature for 12 h. Thecompletion of the reaction was confirmed by TLC analysis. Aftercompletion of reaction it was diluted with DCM and washed with 1.0 M ofsodium bisulphate solution followed by 1.0 M sodium carbonate solution.The organic layer was dried over Na₂SO₄, filtered and evaporated underreduced pressure to yield crude compound 4e, which was further purifiedby silica gel column chromatography (eluent: 0-20% ethyl acetate inhexane) to yield 0.7 g of product 4e. ¹H NMR (DMSO-d⁶, 300 MHz): δ 1.04(s, 9H), 1.16 (d, 3H), 4.11 (m, 1H), 5.11 (m, 3H), 6.91 (d, 2H), 7.40(m, 5H), 8.10 (d, 2H), 8.26 (br s, 1H).

The compounds in Table 3 below were prepared based on the experimentalprocedures described above.

TABLE 3 Compound LCMS HPLC No. Structure (M + H)⁺ t_(R) in min  5.

391.1 12.43  6.

377.1 —  7.

361.1 12.21  8.

230.1 12.95  9.

375.4 11.55 10.

361.2 11.91 11.

361.1 12.08 12.

375.2 11.5  13.

389.1 11.10 14.

347.1 12.58 15.

376.1 12.20 16.

375.2 11.91 17.

361.2 12.34 18.

362.1 12.50 19.

348.1 12.83 20.

391.1 —

The compounds shown in below Table 4, which can be prepared by followingsimilar procedure as described above with suitable modification known tothe one ordinary skilled in the art are also included in the scope ofthe present application.

TABLE 4 Cmpd No. Structure 21.

22.

23.

24.

25.

26.

27.

28.

and 29.

—Rescue of Mouse Splenocyte Proliferation in the Presence of RecombinantPD-L1/PD-L2:

Recombinant mouse PD-L1 (rm-PDL-1, cat no: 1019-B7-100 and R&D Systems)were used as the source of PD-L1.

Requirement:

Mouse splenocytes harvested from 6-8 weeks old C57 BL6 mice; RPMI 1640(GIBCO, Cat #11875); DMEM with high glucose (GIBCO, Cat # D6429); FetalBovine Serum [Hyclone, Cat # SH30071.03]; Penicillin (10000unit/ml)-Streptomycin (10,000 μg/ml) Liquid (GIBCO, Cat #15140-122); MEMSodium Pyruvate solution 100 mM (100×), Liquid (GIBCO, Cat #11360);Nonessential amino acid (GIBCO, Cat #11140); L-Glutamine (GIBCO, Cat#25030); Anti-CD3 antibody (eBiosciences—16-0032); Anti-CD28 antibody(eBiosciences—16-0281); ACK lysis buffer (1 mL) (GIBCO, Cat #-A10492);Histopaque (density—1.083 gm/mL) (SIGMA 10831); Trypan blue solution(SIGMA-T8154); 2 mL Norm Ject Luer Lock syringe-(Sigma 2014-12); 40 μMnylon cell strainer (BD FALCON 35230); Hemacytometer (Bright line-SIGMAZ359629); FACS Buffer (PBS/0.1% BSA): Phosphate Buffered Saline (PBS) pH7.2 (HiMedia TS1006) with 0.1% Bovine Serum Albumin (BSA) (SIGMA A7050)and sodium azide (SIGMA 08591); 5 mM stock solution of CFSE: CFSE stocksolution was prepared by diluting lyophilized CFSE with 180 μL ofDimethyl sulfoxide (DMSO C₂H₆SO, SIGMA-D-5879) and aliquoted in to tubesfor further use. Working concentrations were titrated from 10 μM to 1μM. (eBioscience-650850-85); 0.05% Trypsin and 0.02% EDTA (SIGMA59417C); 96-well format ELISA plates (Corning CLS3390); BD FACS caliber(E6016); Recombinant mouse B7-H1/PDL1 Fc Chimera, (rm-PD-L1 cat no:1019-B7-100).

Protocol

Splenocyte Preparation and Culturing:

Splenocytes harvested in a 50 mL falcon tube by mashing mouse spleen ina 40 μm cell strainer were further treated with 1 mL ACK lysis bufferfor 5 min at room temperature. After washing with 9 mL of RPMI completemedia, cells were re-suspended in 3 mL of 1×PBS in a 15 mL tube. 3 mL ofHistopaque was added carefully to the bottom of the tube withoutdisturbing overlaying splenocyte suspension. After centrifuging at 800×gfor 20 min at room temperature, the opaque layer of splenocytes wascollected carefully without disturbing/mixing the layers. Splenocyteswere washed twice with cold 1×PBS followed by total cell counting usingTrypan Blue exclusion method and used further for cell based assays.

Splenocytes were cultured in RPMI complete media (RPMI+10% fetal bovineserum+1 mM sodium pyruvate+10,000 units/ml penicillin and 10,000 μg/mlstreptomycin) and maintained in a CO₂ incubator with 5% CO₂ at 37° C.

CFSE Proliferation Assay:

CFSE is a dye that passively diffuses into cells and binds tointracellular proteins. 1×10⁶ cells/mL of harvested splenocytes weretreated with 5 μM of CFSE in pre-warmed 1×PBS/0.1% BSA solution for 10min at 37° C. Excess CFSE was quenched using 5 volumes of ice-coldculture media to the cells and incubated on ice for 5 min. CFSE labelledsplenocytes were further given three washes with ice cold complete RPMImedia. CFSE labelled 1×10⁵ splenocytes added to wells containing eitherMDA-MB231 cells (1×10⁵ cells cultured in high glucose DMEM medium) orrecombinant human PDL-1 (100 ng/mL) and test compounds. Splenocytes werestimulated with anti-mouse CD3 and anti-mouse CD28 antibody (1 μg/mLeach), and the culture was further incubated for 72 h at 37° C. with 5%CO₂. Cells were harvested and washed thrice with ice cold FACS bufferand % proliferation was analyzed by flow cytometry with 488 nmexcitation and 521 nm emission filters.

Data Compilation, Processing and Inference:

Percent splenocyte proliferation was analyzed using cell quest FACSprogram and percent rescue of splenocyte proliferation by compound wasestimated after deduction of % background proliferation value andnormalising to % stimulated splenocyte proliferation (positive control)as 100%.

Stimulated splenocytes: Splenocytes+anti-CD3/CD28 stimulation

Background proliferation: Splenocytes+anti-CD3/CD28+PD-L1

Compound proliferation: Splenocytes+anti-CD3/CD28+PD-L1+Compound

Compound effect is examined by adding required concentration of compoundto anti-CD3/CD28 stimulated splenocytes in presence of ligand (PDL-1).

TABLE 5 Percent rescue of splenocyte proliferation Compound (@100 nMcompound No. concentration)  1 61.2  2 80.3  3 48.4  4 60  9 74 10 58 1292 13 75 14 53 15 69 16 56 17 53 18 68 — —

What is claimed is:
 1. A method of making a compound according to thefollowing scheme:

wherein: R₁ represents —CH₂OH, —CH(CH₃)OH, —CH₂Ph, —CH₃, or —CH₂C(O)NH₂optionally substituted with alkyl; X is S or O; Aaa is an amino acidresidue selected from Ser, Asn or Thr, optionally substituted by alkyl;wherein the C-terminus thereof is a free terminus, is amidated, or isesterified; and R₃ represents —CH₂OH, —CH₃, —CH₂CH₂C(O)OH,—CH₂CH₂C(O)NH₂, —CH₂C(O)NH₂, or —CH₂C(O)OH, wherein each —CH₂CH₂C(O)OH,—CH₂CH₂C(O)NH₂, —CH₂C(O)NH₂, or —CH₂C(O)OH is optionally substitutedwith alkyl or aralkyl.
 2. The method of claim 1, wherein Aaa is an aminoacid residue selected from Thr and Ser, optionally substituted by alkyl;wherein the C-terminus thereof is a free terminus or is esterified. 3.The method of claim 2, wherein Aaa is an amino acid residue selectedfrom Thr and Ser substituted by alkyl; wherein the C-terminus thereof isesterified.
 4. The method of claim 1, wherein R₁ represents —CH₂OH or—CH(CH₃)OH, optionally substituted with alkyl.
 5. The method of claim 1,wherein X is S.
 6. The method of claim 1, wherein X is O.
 7. The methodof claim 1, wherein R₃ represents —CH₃, —CH₂CH₂C(O)OH, —CH₂CH₂C(O)NH₂,—CH₂C(O)NH₂, or —CH₂C(O)OH, wherein each —CH₂CH₂C(O)OH, —CH₂CH₂C(O)NH₂,—CH₂C(O)NH₂, or —CH₂C(O)OH is optionally substituted with alkyl oraralkyl.
 8. The method of claim 1, wherein the base is triethylamine. 9.The method of claim 1, wherein the solvent is tetrahydrofuran ordimethyl formamide.
 10. The method of claim 1, wherein: X is S or O, Aaais an amino acid residue selected from Ser, Asn or Thr, substituted byalkyl; wherein the C-terminus thereof is a free terminus or isesterified; R₁ represents —CH₂OH, —CH(CH₃)OH, —CH₂Ph, —CH₃, or—CH₂C(O)NH₂, wherein each —CH₂OH, —CH(CH₃)OH, or —CH₂C(O)NH₂ isoptionally substituted with alkyl; and R₃ represents —CH₂OH, —CH₃,—CH₂CH₂C(O)OH, —CH₂CH₂C(O)NH₂, —CH₂C(O)NH₂, or —CH₂C(O)OH, wherein each—CH₂CH₂C(O)OH, —CH₂CH₂C(O)NH₂, —CH₂C(O)NH₂, or —CH₂C(O)OH is optionallysubstituted with alkyl or aralkyl; further comprising the step ofcontacting the compound of formula

with an acid, to form a compound of formula

wherein: Aaa′ is an amino acid residue selected from Ser, Asn or Thr;wherein the C-terminus thereof is a free terminus; R_(1a) represents—CH₂OH, —CH(CH₃)OH, —CH₂Ph, —CH₃, or —CH₂C(O)NH₂, wherein each —CH₂OH,—CH(CH₃)OH, or —CH₂C(O)NH₂ is optionally substituted with alkyl; andR_(1a) represents —CH₂OH, —CH₃, —CH₂CH₂C(O)OH, —CH₂CH₂C(O)NH₂,—CH₂C(O)NH₂, or —CH₂C(O)OH, wherein each —CH₂CH₂C(O)OH, —CH₂CH₂C(O)NH₂,—CH₂C(O)NH₂, or —CH₂C(O)OH is optionally substituted with alkyl oraralkyl.